Heart disease is leading cause of death in the United States. High-density lipoprotein (HDL) levels are inversely correlated with the prevalence of coronary heart disease. The anti-atherogenic properties of HDL are associated with its role in the pathway of the reverse cholesterol transport, which removes cholesterol from peripheral cells for transport back to hepatocytes. The formation of HDL is facilitated by ATP cassette transporter ABCA1 and apolipoprotein A-I, the major protein of HDL particles. However, the underlying molecular mechanism behind the biogenesis of HDL is not well understood. To provide further understanding of this mechanism, we developed two ABCA1 expression systems in both Sf9 insect cells and FreestyleTM 293-F human cells for functional and structural studies. We designed all constructs of ABCA1 to contain a C-terminal rho1d4 tag that bound to an affinity matrix of rho1d4 antibodies for successful purification. To reconstitute ABCA1 in a detergent-free environment that models the native membrane, we developed three reconstitution systems for ABCA1: saposin A nanodiscs, peptidisc, and amphipol A8-35. Biochemical and structural studies were carried out to understand the mechanism behind ABCA1’s function. We demonstrated a potential direct interaction of ABCA1 and apolipoprotein A-I with a pull-down experiment. Two cryo-electron microscopy data collections were obtained of ABCA1 in a detergent environment in the presence of ATP with the goal of determining the structure of ABCA1’s active state. We produced a 12 Å reconstruction of ABCA1 from this first data collection. This low-resolution structure confirmed the general structure that currently exists for ABCA1. Processing the data helped us streamline and troubleshoot the electron microscopy workflow pipeline for future data collections. Unfortunately, the second data collection had astigmatism issues that prevented particle alignment during data analysis. However, these data collections provided considerable insight into the ideal sample freezing and grid preparation conditions that affect data collection and data processing. More transmembrane protein structures are being solved each year but there remain many obstacles and challenges in ABCA1 purification and grid preparation that affect the ability to perform functional studies and high-resolution structure determination. Our developmental work has helped move forward our biochemical understanding of ABCA1 to achieve these aims. The more that is learned about this important membrane protein the more likely it is that future consistent production of ABCA1 will be accomplished to answer the questions of how ABCA1 mediates the formation of HDL particles.

Tubulin and elastin-like polypeptides (ELPs) both form large protein structures which can be thermodynamically evaluated using isothermal titration calorimetry and differential scanning calorimetry. ELPs are thermos-responsive biopolymers that undergo phase separation and form coacervates when heated. This project assesses the liquid-liquid phase separation of an ELP sequence derived from tropoelastin with a SynB1 cell-penetrating peptide attached to the N-terminus in conjunction with the chemotherapeutic drug doxorubicin. Microtubules (MTs) are a dynamic cellular structure formed of tubulin alpha/beta-heterodimers and are responsible for several important cellular processes, making them a viable target for anti-cancer drugs. There has been extensive research done to identify new ligands that show selective binding to microtubules. Ruthenium (II) polypyridyl complexes (RPCs) have been found to promote the polymerization of tubulin into microtubules. ITC has been used to determine the binding affinity of [Ru(II)(Ph2phen)3]2+ (RPC2).

No description available.

No description available.

Gene regulation is a critical aspect of normal development, energy conservation, metabolic control, and responses to environmental cues, diseases and pathogens in eukaryotic organisms. In order to appropriately respond to environmental changes and advance through the life cycle, an organism must manage the expression levels of a large number of genes by utilizing available gene regulation mechanisms. The developmental control of 5â -nucleotidase (5nt) expression in the model system Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. In order to identify temporally-regulated control elements within the promoter of the 5nt gene, 5â and internal promoter deletions were designed and fused to the luciferase and lacZ reporter genes, and reporter enzyme activity was measured in cells from the slug stage of development. The results from these experiments enabled the identification of a 250 bp region of the promoter, which was used as a template for subsequent site-directed mutagenesis experiments. These experiments involved altering 6-12 bp regions of the promoter by substitution. Twelve mutagenized promoters were fused to the luciferase and lacZ reporter genes, and activity was measured at the slug stage of development to more precisely locate cis-acting temporally-regulated control elements. In addition, cAMP induction experiments were performed on amoebae transformed with the mutagenized promoters to identify control elements within the promoter influenced by the presence of cAMP. The regions between -530 and -560 bp and -440 and -460 bp from the ATG translation start site. In order to evaluate the functions of the cis-acting promoter control elements, electromobility gel shift assays were performed to identify specific DNA-protein interactions on the 5nt promoter. These assays enabled the detection of a 0.13 Rf and 0.33 Rf binding activity to specific sites of the promoter. After characterization of these binding activities, both proteins were purified by a series of column chromatography techniques and characterized after mass spectrometry. The proteins purified were identified as formyltetrahydrofolate synthase and hydroxymethylpterin pyrophosphokinase. These enzymes function in the biosynthetic pathway of tetrahydrofolate and the production of folate coenzymes. The specific interactions of these enzymes with the 5nt promoter suggest these proteins may also function in regulating 5nt expression. / Ph. D.

Regulation of gene expression is known to be a critical factor involved in proper development, responses to environmental cues, metabolism, energy conservation, and disease. Gene expression is regulated at several levels including transcription, mRNA splicing, post translational modification, and the rate of protein degradation. The developmental control of <i>alkaline phosphatase</i> (alp) in <i>Dicytostelium</i> has provided a focal point for the study of gene regulation at the level of <i>de novo</i> synthesis. The localization of <i>alkaline phosphatase</i> (alp) expression during development was characterized by fusing the 5' flanking sequence to the <i>lacZ</i> reporter and using an <i>in situ</i> β-galactosidase staining method. The localization of </i>lacZ</i> expression corresponds with that of the endogenous ALP enzyme suggesting that <i>alp</i> is regulated at the level of transcription. In order to identify temporal regulatory elements within the <i>alp</i> promoter a series of 5' and internal promoter deletions were generated and fused to the <i>lacZ</i> reporter. The data from these promoter deletion constructs indicated a regulatory element within the -683 to -468 bp sequence that is required for normal expression of <i>alp</i> during development. A series of small internal and 5' promoter deletions were designed within the -683 to -468 bp regulatory sequence. The results from these promoter deletion-reporter gene fusions suggested a DNA regulatory element is located within a 26-bp sequence beginning at the -620 bp site. The function of <i>cis</i>-acting regulatory elements were evaluated using the electromobility shift assay (EMSA) to identify sequence specific DNA-protein interactions on the <i>alp promoter</i>. We report the characterization of three DNA-binding activities with the 20% ammonium sulfate (AS) slug nuclear fraction. These DNA-binding activities appear to be related as they all require magnesium or calcium for effective binding to the <i>alp</i> promoter. Interestingly, the DNA-binding proteins appeared to interact with a GT-rich sequence that contained a G-box binding factor (GBF) consensus element. Additionally, a DNA-binding activity observed in the 80% AS slug nuclear extract was characterized and sequentially purified using conventional and affinity chromatography techniques. The DNA-binding protein was identified as TFII, a protein that was previously identified during the investigation of <i>glycogen phosphorylase-2 (gp2)</i> regulation. A comparison of the <i>alp</i> and <i>gp2</i> probes used to identify TFII suggests a DNA-binding site, ACAATGN₈₋₁₂CACTA. The ability of TFII to bind specifically with the promoter of two functionally different genes suggests that it may regulate the temporal and/or spatial expression of several <i>Dictyostelium</i> genes. / Ph. D.

Erythropoietin is a human natural hormone which task is to regulate the amount of red blood cells in the body. At Centro de Inmunología Molecular, situated in Havana, erythropoietin is produced by recombinant DNA-technique. The protein is purified through several chromatography steps. Among other things, Centro de Inmunología Molecular uses affinity chromatography and ion exchange chromatography. To both of these chromatographic methods, gel is used as stationary phase. The aim of this study was to investigate and determine parameters for characterising of two gels, this because Centro de Inmunología Molecular have to exchange the gels. The reason for the gel exchange is that the currently used gels will not be manufactured any more. The gel used in the affinity chromatography is Chelating Sepharose Fast Flow and the gel used in the ion exchange chromatography is Q Sepharose Fast Flow. For both of this gels kinetic parameters and isotherm parameters were determined by experiments. The isotherm parameters qmax and Kd were calculated from an adsorption isotherm. To be able to calculate qmax and Kd for both Q Sepharose Fast Flow gel and Chelating Sepharose Fast Flow gel different experiments were made. A kinetic adsorption and an isotherm adsorption were made on each gel. The kinetic adsorption was made in due to find out how long the two different processes were supposed to run and to understand which part of the mass transfer that is controlling the rate. There is no use to let the process to be in progress any longer than until the adsorption ceases. For the Q Sepharose Fast Flow gel this was after 200 seconds. The adsorption with the Chelating Sepharose Fast Flow gel never ceased completely, but after 1000 seconds the adsorption was so slow that it would be no use to continue the process. If the processes continue after the calculated times only money, hours and recourses will be wasted. The data that were achieved was plotted in two different isotherm adsorption models both the Freundlich- and the Langmuir model, this to determine which model that had the best fit. One could see that the Q Sepharose Fast Flow gel was following the model of Langmuir better and because of this the Langmuir equation was used to calculate qmax and Kd. The qmax for the Q Sepharose Fast Flow gel agreed a lot with the value that Centro de Inmunología Molecular had assumed. When it came to the Chelating Sepharose Fast Flow gel, the same kind of plotting was made. But one could see that this time the data was following the model of Freundlich much better. Therefore a calculation of the desired qmax was impossible. Only the value of Kd was calculated. Because the company Centro de Inmunología Molecular still needed the value of qmax an assumption that the gel was following the model of Langmuir was made. qmax was calculated but without any satisfied results. The programs Excel, Statgraphic and Matlab have been used in all calculations. / Uppsatsnivå: C

Mutations in the human LMNA gene encoding A-type lamins cause a collection of diseases termed laminopathies, including several types of muscular dystrophy. Lamins are intermediate filaments, which line the inner membrane of nuclear envelope. Lamins maintain the nuclear shape and regulate gene expression through interactions with chromatin. Heterozygous mutations LMNA, which result in single amino acid substitutions within the C-terminal Ig-fold domain, were identified in patients with muscular dystrophy. These substitutions were modeled in Drosophila and found to cause muscle defects. We have taken a multi-disciplinary approach to understanding the molecular basis of these muscle defects. Using Nuclear Magentic Resonance (NMR) and Circular Dischroism (CD) we determined that the amino acid substitutions cause perturbations of the tertiary, but not secondary, structure of the Ig-fold. Microarray analysis of RNA isolated from muscle revealed that mutant lamins cause cause mis-regulation of genes involved oxidative stress and neuromuscular junction function. Collectively, these data demonstrate that perturbations within the lamin Ig-fold cause changes in gene expression, providing insights on pathways involved in pathogenesis and identifying new potential therapeutic targets.

Single particle electron cryomicroscopy (cryo-EM) is a rapidly developing structural biology technique for the study of macromolecular protein complexes. Presently, cryo-EM fills an important niche by facilitating acquisition of 3-D structures of protein complexes not amenable to structure determination by other techniques. Expansion of cryo-EM beyond this niche requires continued improvement in the types of specimens that can be studied as well as the final resolutions achieved. Two studies were undertaken to address these issues. The first examined resolution limitations by quantifying the effect of beam-induced motion in images of beam-sensitive paraffin crystals. The second explored the possibility of using cryo-EM to study the interaction of small effector proteins with a large multi-protein complex, V-ATPase. The results of these studies exposed the fact that fundamental aspects of the imaging and specimen preparation processes remain poorly understood and must be addressed to facilitate future improvements in cryo-EM structure determination.

Protein arginine methyltransferases (PRMTs)mediate the transfer of methyl groups to arginine residues in histone and non-histone proteins. PRMT5 is an important member of PRMTs which symmetrically dimethylates arginine 8 in histone H3 (H3R8) and arginine 3 in histone H4 (H4R3). PRMT5 was reported to inhibit some tumor suppressors in leukemia and lymphoma cells and regulate p53 gene, through affecting the promoter of p53. Through methylation of H4R3, PRMT5 can recruit DNA-methyltransferase 3A (DNMT3A) which regulates gene transcription. All the above suggest that PRMT5 has an important function of suppressing cell apoptosis and is a potential anticancer target. Currently, the enzymatic activities of PRMT5 are not clearly understood. In our study, we improved the protein expression methodology and greatly enhanced the yield and quality of the recombinant PRMT5. In addition, mutagenesis and enzymatic studies implicate an interesting mechanism of PRMT5 activity regulation.